1. Field of the Invention
Significant morbidity and mortality are associated with infectious diseases. More rapid and accurate diagnostic methods are required for better monitoring and treatment of disease. Molecular methods using DNA probes, nucleic acid hybridizations and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the 32P labeling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient""s body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
When very low concentrations must be detected, the above method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable.
Recently, a method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the 5xe2x80x2 ends of the oligonucleotide primers.
Other methods for amplifying nucleic acids have also been developed. These methods include single primer amplification, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and the Q-beta-replicase method. Regardless of the amplification used, the amplified product must be detected.
After amplification of a particular nucleic acid, a separate step is carried out prior to detecting amplified material. The various nucleic acid amplification procedures developed over the past few years greatly enhanced the sensitivity of detecting defined nucleic acid species in a test sample. The frequent formation of non-specific amplification products requires selective detection of the specific amplification product, which is often carried out using multiple probes complementary to regions within the specific amplified sequence.
One method for detecting nucleic acids is to employ nucleic acid probes that have sequences complementary to sequences in the amplified nucleic acid. One method utilizing such probes is described in U.S. Pat. No. 4,868,104. A nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support. Detection of signal depends upon the nature of the label or reporter group. If the label or reporter group is an enzyme, additional members of the signal producing system include enzyme substrates and so forth.
Usually, the probe is comprised of natural nucleotides such as ribonucleotides and deoxyribonucleotides and their derivatives although unnatural nucleotide mimetics such as peptide nucleic acids (PNA) and oligomeric nucleoside phosphonates are also used. Commonly, binding of the probes to the target is detected by means of a label incorporated into the probe. Binding can be detected by separating the bound probe from the free probe and detecting the label. For this purpose it is usually necessary to form a sandwich comprised of the labeled probe, the target and a probe that is or can become bound to a surface. Alternatively, binding can be detected by a change in the signal-producing properties of the label upon binding, such as a change in the emission efficiency of a fluorescent or chemiluminescent label. This permits detection to be carried out without a separation step.
Homogeneous methods that have been utilized include the Taqman method used by Roche Molecular Diagnostics. In this approach a probe is used that is labeled with a fluorescer and a quencher. The polymerase used in PCR is capable of cutting the probe when it is bound to the target DNA and causing separation of these labels. Changes in the polarization of fluorescence upon binding of a fluorescer-labeled probe to target DNA are used by Becton Dickenson to detect the formation of DNA in Strand Displacement Amplification (SDA). Binding of two probes, one with a chemiluminescer bead and one with a sensitizer bead has been used by Behring Diagnostics Inc. for detection of DNA produced by PCR and single primer amplification. Binding of an electroluminescent ruthenium labeled probe to a biotinylated target RNA and capture of the complex on magnetic beads has been used by Organon Teknika for detection of RNA produced in NASBA. GenProbe has carried out detection of RNA by means of an acridinium labeled probe that changes chemiluminescence efficiency when the probe is bound to target RNA.
Each of the above methods has limitations. Where two or more probes are required for detection and quantitation of products of specific nucleic acid amplifications, increasing the amount of target increases the signal up to a point and then the signal falls off (the xe2x80x9cprozonexe2x80x9d phenomenon). In general, loss of signal is realized under the prozone phenomenon when the analyte concentration exceeds probe concentration. Methods that require a capturable ligand in the target cannot be used on non-amplified nucleic acids nor are all amplification methods capable of introducing a ligand into the amplified product. Fluorescence polarization changes on binding are small and the sensitivity is therefore limited. Taqman is subject to problems with emission from the quencher, which limits sensitivity; GenProbe""s chemiluminescent probe requires reagent additions prior to detection.
It is desirable to have a sensitive, simple method for amplifying and detecting nucleic acids preferably, in a homogeneous format. The method should minimize the number and complexity of steps and reagents and avoid the prozone phenomenon.
Also, it is desirable to know the concentration of the amplified product in the reaction medium. Quantitation of nucleic acid amplification products has become an important molecular diagnostic tool.
2. Description of the Related Art
Rapid, non-separation electrochemiluminescent DNA hybridization assays for PCR products using 3xe2x80x2-labeled oligonucleotide probes is described by Gudibande, et al., (1992) Molecular and Cellular Probes, 6: 495-503. A related disclosure is found in international patent application WO 9508644 A1 (950330).
Marmaro, et al., (Meeting of the American Association of Clinical Chemists, San Diego, Calif., November 1994, Poster No. 54) discusses the design and use of fluorogenic probes in TaqMan, a homogeneous PCR assay.
German patent application DE 4234086-A1 (92.02.05) (Henco, et al.,) discusses the determination of nucleic acid sequences amplified in vitro in enclosed reaction zone where probe(s) capable of interacting with target sequence is present during or after amplification and spectroscopically measurable parameters of probe undergo change thereby generating signal.
A process for the determination of nucleic acid molecules at low concentrations by amplification using labeled primers is discussed in patent application WO 96-EP5472 961206 (Eigen, et al.).
A self-sustained sequence replication electrochemiluminescent nucleic acid assay is disclosed in patent application WO 94-US10732 940921 (Kenten, et al.).
A hybridization protection assay is discussed by Matsuoka in Rinsho Kensa (1991) 35(6):627-631.
U.S. Pat. No. 5,593,867 (Walker, et al.) discloses a fluorescence polarization detection of nucleic acid amplification using fluorescently labeled oligonucleotide probes and detector-probe extension products.
Molecular beacons: probes that fluoresce upon hybridization are discussed by Tyagi, et al.) in Nat. Biotechnol. (1996) 14(3):303-308.
U.S. Pat. No. 5,656,207 (Woodhead, et al.) discloses a method for detecting or quantifying multiple analytes using labelling techniques.
U.S. Pat. No. 5,340,716 (Ullman, et al.) describes an assay method utilizing photoactivated chemiluminescent labels.
Photoactivatable chemiluminescent matrices are described in patent application WO 94/03812 (Pease, et al.).
European Patent Application No. 0 515 194 A2 (Ullman, et al.) discloses assay methods utilizing induced luminescence. The references cited therein are incorporated herein by reference including without limitation U.S. Pat. No. 5,017,473 (Wagner), which discloses a homogeneous chemiluminescence immunoassay using a light absorbing material, European Patent Application No. 0,345,776 (McCapra), which discloses specific binding assays that utilize a sensitizer as a label, U.S. Pat. No. 4,193,983 (Ullman, et al.), which discloses labeled liposome particle compositions and immunoassays therewith, U.S. Pat. No. 4,891,324 (Pease, et al.), which describes a particle with luminescer for assays.
One embodiment of the present invention is a method for detecting the amount of a target polynucleotide in a sample. A combination is provided in a medium. The combination comprises (i) a sample suspected of containing the target polynucleotide, the target polynucleotide being in single stranded form, (ii) a reference polynucleotide comprising a sequence that is common with a sequence of the target polynucleotide, and (iii) a predetermined amount of an oligonucleotide probe that has a sequence that hybridizes with the sequence that is common. The combination is subjected to conditions for amplifying the target polynucleotide and the reference polynucleotide. The conditions permit formation of substantially non-dissociative complexes of the target polynucleotide and the reference polynucleotide, respectively, with the oligonucleotide probe. Furthermore, the predetermined amount of the oligonucleotide probe is less than the expected amount of the amplified target polynucleotide. The ratio of the amount of the complex of the target polynucleotide with the oligonucleotide probe to the amount of the complex of the reference polynucleotide with the oligonucleotide probe is determined. The ratio is related to the known amount of the reference polynucleotide to determine the amount of the target polynucleotide in the sample.
Another aspect of the present invention concerns a method for detecting the amount of a target polynucleotide in a sample. In this aspect a combination is provided in a medium. The combination comprises (i) a sample suspected of containing the target polynucleotide, the target polynucleotide being in single stranded form, (ii) predetermined amounts of one or more reference polynucleotides, each of the reference polynucleotides comprising a first sequence that is common with a first sequence of the target polynucleotide and a second sequence that is different from a second sequence of the target polynucleotide, (iii) a predetermined amount of a first oligonucleotide probe that has a sequence that hybridizes with the sequence that is common, (iv) a second oligonucleotide probe that has a sequence that hybridizes only with the second sequence of the target polynucleotide, and (v) one or more third oligonucleotide probes. Each of the third oligonucleotide probes has a sequence that hybridizes only with a respective second sequence of one of the reference polynucleotide. The combination is subjected to isothermal conditions for amplifying with equal efficiency the target polynucleotide and the one or more reference polynucleotides. The conditions permit formation of a substantially non-dissociative first termolecular complex of the target polynucleotide, the first oligonucleotide probe and the second oligonucleotide probe and a substantially non-dissociative second termolecular complex of each of the reference polynucleotide with the first oligonucleotide probe and a respective third oligonucleotide probe. The predetermined amount of the first oligonucleotide probe is less than the expected amount of the amplified target polynucleotide. A determination is made of the ratio of the amount of the first termolecular complex to the amount of each of the second termolecular complexes. Each of the ratios is related to the predetermined amount of each of the reference polynucleotides to determine the amount of the target polynucleotide in the sample.
Another embodiment of the present invention is a method for detecting the amount of a target polynucleotide in a sample. In this embodiment a combination is provided in a medium. The combination comprises (i) a sample suspected of containing the target polynucleotide, the target polynucleotide being in single stranded form, (ii) predetermined amounts of one or more reference polynucleotides, each of the reference polynucleotides comprising a first sequence that is common with a first sequence of the target polynucleotide and a second sequence that is different from a second sequence of the target polynucleotide, (iii) a predetermined amount of a first oligonucleotide probe that has a sequence that hybridizes with the sequence that is common wherein the first oligonucleotide probe has, or is capable of having, a sensitizer attached thereto, (iv) a second oligonucleotide probe that has a sequence that hybridizes only with the second sequence of the target polynucleotide wherein the second oligonucleotide probe has, or is capable of having, a first chemiluminescent compound attached thereto, and (v) one or more third oligonucleotide probes. Each of the one or more third oligonucleotide probes has a sequence that hybridizes only with a respective second sequence of one of the reference polynucleotide. In addition, each of the third oligonucleotide probes has, or is capable of having, a second chemiluminescent compound attached thereto. The first and the second chemiluminescent compounds differ in signal produced when activated by the photosensitizer. The second chemiluminescent compound is different for each of the third oligonucleotide probes. The combination is subjected to isothermal conditions for amplifying with equal efficiency the target polynucleotide and each of the reference polynucleotide. The conditions permit formation of a substantially non-dissociative first termolecular complex of the target polynucleotide, the first oligonucleotide probe and the second oligonucleotide probe and a substantially non-dissociative second termolecular complex of each of the reference polynucleotides with the first oligonucleotide probe and a respective third oligonucleotide probe. The predetermined amount of the first oligonucleotide probe is less than the expected amount of the amplified target polynucleotide. The ratio of the amount of the signal produced by the first termolecular complex to the amount of signal produced by each of the second termolecular complexes is determined. Each of the ratios is related to the amount of each respective reference polynucleotide to determine the amount of the target polynucleotide in the sample.
Another embodiment of the present invention is a kit for use in amplification and detection of a target polynucleotide. The kit is a packaged combination and comprises reagents for conducting an amplification of the target polynucleotide and predetermined amounts of one or more reference polynucleotides. Each of the reference polynucleotides comprises a first sequence that is common with a first sequence of the target polynucleotide and a second sequence that is different from a second sequence of the target polynucleotide. Also in the kit are a predetermined amount of a first oligonucleotide probe that has a sequence that hybridizes with the sequence that is common and a second oligonucleotide probe that has a sequence that hybridizes only with the second sequence of the target polynucleotide. The kit further comprises one or more third oligonucleotide probes, each of the third oligonucleotide probes having a sequence that hybridizes only with a respective second sequence of one of the reference polynucleotide. The kit may also comprise reagents for conducting an isothermal amplification.
Another aspect of the present invention is a kit for use in an amplification and quantitation of a specific RNA. The kit comprises in packaged combination one or more reference RNA""s, a promoter, an enzyme and a predetermined amounts of one or more reference polynucleotides. Each of the reference polynucleotides comprising a first sequence that is common with a first sequence of the target polynucleotide and a second sequence that is different from a second sequence of the target polynucleotide. The kit also comprises a predetermined amount of a first oligonucleotide probe that has a sequence that hybridizes with the sequence that is common. The first oligonucleotide probe is labeled with a sensitizer. A second oligonucleotide probe is included that has a sequence that hybridizes only with the second sequence of the target polynucleotide. The second oligonucleotide probe is labeled with a chemiluminescer. The kit further comprises one or more third oligonucleotide probes. Each of the third oligonucleotide probes has a sequence that hybridizes only with a respective second sequence of one of the reference polynucleotide. In addition, each of the third oligonucleotide probes is labeled with a chemiluminescer where the chemiluminescer is different from that of the second oligonucleotide probe and different for each of the third oligonucleotide probes.